The present invention relates to Semaphorin Z, a novel Semaphorin belonging to the Semaphorin family, and use of Semaphorin Z for pharmaceutical agents or laboratory reagents. More particularly, it relates to Semaphorin Z inhibiting neurite outgrowth, and a gene encoding the same, as well as other Semaphorins hybridizing to said Semaphorin Z gene. Furthermore, the present invention relates to modified proteins or partial peptides of Semaphorin Z, antibodies against Semaphorin Z, DNAs or RNAs complementary to said Semaphorin Z gene, and their use for pharmaceutical or diagnostic agents or laboratory reagents.
It is widely known that a central nervous system (CNS)-neuron in higher organisms such as human is not capable of regeneration once injured. Therefore, one who has received an injury on his (her) spinal cord due to, for example, a traffic accident is compelled to spend the rest of his (her) life in a hemiplegic state. On the contrary, it is known that a peripheral nervous system (PNS)-neuron retains a vigorous regeneration ability even in those higher organisms, and therefore, neurons in a limb, when disconnected, can gradually regenerate with a concomitant recovery of their function.
In early nineteen-eighties, a group of Aguayo et al. found that when PNS-neuron is experimentally grafted into an injured CNS-neuron in a higher organism, axon growth of CNS-neuron is induced. This observation demonstrates that CNS-neuron in higher organisms which had been generally considered not to have a regeneration ability can regenerate if a suitable environment is provided (Nature, 284, 264-265 (1980), Science, 214, 931-933 (1981)). That report suggests a possibility that in CNS of higher organisms, there may exist a factor, namable xe2x80x9cCNS-neuron regeneration inhibitorxe2x80x9d, which inhibits the regeneration of CNS-neuron, and that a release from such inhibition may allow the regeneration of CNS-neurons. This suggestion paved the way for a CNS-neuron regeneration therapy.
In 1988, a group of Schwab et al. demonstrated that there existed such CNS-neuron regeneration inhibitor among proteins derived from CNS myelin. They also succeeded in purifying, though partially, a protein having said CNS-neuron regeneration inhibition activity, and named this protein fraction NI35/250 (Annu. Rev. Neurosci., 16, 565-595 (1993)), although no one has succeeded in its isolation, identification and gene cloning yet. In addition, they immunized animals with the partial purified NI35/250, and succeeded in obtaining an antibody (IN-1) having a neutralizing activity. This antibody is capable of recognizing a band for NI35/250 in Western blotting, and capable of staining, in an immunostaining, the region where NI35/250 is supposed to be distributed. Furthermore, they demonstrated that administration of this antibody to an animal experimentally received an injury on its spinal cord has promoted regeneration of axons in spinal cord, though partially, within 2-3 weeks, and restored its function within 2-3 months (Nature, 343, 269-272 (1990), Nature, 378, 498-501 (1995)). These findings are of great value, because they experimentally demonstrated that there existed a CNS-neuron regeneration inhibitor as suggested by Aguayo et al. (supra) and that CNS-neuron can be regenerated by inhibiting the activity of said inhibitor. The above noted antibody is, however, directed not to human but to rat NI35/250, and exhibits a low stability and specificity. In addition, although regeneration of CNS-neuron was observed as described above by administering said antibody, its effect was so partial and incomplete that not all of the motor functions could be restored. It is, therefore, believed essential in solving these problems to identify the gene coding for NI35/250 or corresponding CNS-neuron regeneration inhibitor, and, based on knowledges of molecular biology, neuroscience and the like, develop an inhibitor effectively inhibiting the CNS-neuron regeneration inhibition activity, or develop a method for inhibiting the expression of the gene for said regeneration inhibitor.
Apart from the above, the nervous system, whether it is central or peripheral, requires formation of a complicated neural network among neurons or between neurons and peripheral receivers or effectors during development, that is, in the stage of embryo or fetus, in order to precisely carry out its principal functions, i.e., to transfer and process the information. To establish the neural network, an ingenious mechanism is necessary, which precisely guides a growing neurite to the target site locating remote therefrom.
It has been hitherto believed that a factor which positively control the neurite outgrowth such as neurite growth promoter and neurite growth attractant may play a major role in the formation of the neural network. However, it is now being demonstrated by recent studies on the mechanism of the network formation that the opposite factor, that is, a negative factor having an outgrowth inhibition activity is important for an accurate guidance (Cell, 78, 353-356 (1994)).
A representative factor having such an outgrowth inhibition activity is a protein called xe2x80x9cSemaphorinxe2x80x9d. Semaphorin firstly discovered is Fasciclin IV found in grasshopper. Collapsin (latterly named Collapsin I) was subsequently discovered in chick (Cell, 75, 217-227 (1993); Neuron, 9, 831-845 (1992)). To date, more than 10 genes belonging to the Semaphorin family have been reported in a wide range of species covering insects such as drosophila and beetle, human, and viruses (Cell, 81, 471-474 (1995)). These Semaphorins characteristically contains in their amino acid sequences a certain structure called semaphorin domain consisting of about 500 amino acids (Neuron, 14, 941-948 (1995); Cell, 75, 1389-1399 (1993)). However, the homologies of the primary amino acid sequences in Semaphorin domains among these Semaphorin genes are 80-20%, and not necessarily high.
Of these Semaphorins, functions have been verified for only a few, including, for example, Fasciclin IV of grasshopper, Semaphorins I and II of drosophila, Collapsin of chick, and Semaphorin III which corresponds to Collapsin in mammals. All of these Semaphorins are known to inhibit neurite outgrowth and synapsis formation. In particular, Semaphorin III has been reported to have an activity collapsing in a short time the growth cone of cultured neuron (growth-cone collapse activity) in vitro (Neuron, 14, 941-948 (1995); Neuron, 14, 949-959 (1995); Cell, 81, 631-639 (1995); Cell, 75, 1389-1399 (1993); Cell, 75, 217-227 (1993); Neuron, 9, 831-845 (1992)).
Although it is now being demonstrated, as described above, that Semaphorin has a growth-cone collapse activity and a neurite outgrowth inhibition activity during development, and plays a role in giving an accurate guidance to neuron, it is not evident at present whether or not Semaphorin exerts some function not only during development but also in the adult, and less evident whether or not Semaphorin plays a role as a CNS-neuron regeneration inhibitor. Of course, since Semaphorin has been shown to be a negative guidance factor inhibiting neurite outgrowth, it would not be unreasonable to consider said Semaphorin as a candidate for a CNS-neuron regeneration inhibitor (Nature, 378, 439-440 (1995)). However, it has been shown by in vitro experiments that Semaphorin III (Sema III), only one Semaphorin of higher organisms of which function has been analyzed, exerts its neurite-outgrowth inhibition activity on a sensory neuron and sympathetic neuron both of which are peripheral, but not on a retinal neuron which is central (Cell, 75, 217-227 (1993)). In addition, Northern analysis on the distribution of Sema III expression in the adult conducted by the present inventors has revealed that it is expressed mainly in peripheral tissues (see Reference example 2 below). It is therefore hardly believed that Sema III having such features has a function as a xe2x80x9cCNS-neuron regeneration inhibitorxe2x80x9d.
The present invention aims to provide Semaphorin Z, a novel Semaphorin inhibiting neurite outgrowth, and a gene therefor, and to provide a pharmaceutical or diagnostic agent for neural diseases, in particular an agent for regeneration of CNS-neuron, as well as a laboratory reagent. Based on the discovery of Semaphorin Z, the present invention also provides, for example, another Semaphorin gene hybridizing to said Semaphorin Z gene, a modified Semaphorin Z protein or a partial peptide of Semaphorin Z, an antibody against Semaphorin Z, DNA or RNA complementary to said Semaphorin Z gene, a screening method for Semaphorin Z inhibitor using Semaphorin Z, a Semaphorin Z inhibitor obtained by said screening method, a pharmaceutical composition comprising Semaphorin Z or an inhibitor thereof, a transgenic animal involving Semaphorin Z. Furthermore, the present invention provides a laboratory reagent for this technical field on the basis of the discovery of Semaphorin Z.
If regeneration of CNS-neuron in the adult is always kept inhibited as described in the xe2x80x9cPrior Artxe2x80x9d section, it is believed that identification of a factor which inhibits regeneration of CNS-neuron is the most important subject to be solved for establishing a therapy for regeneration of CNS-neuron, and that any therapy for regeneration of CNS-neuron can not be established without identifying such factor.
The present inventors have paid their attention to the similarity between the in vitro activities of the above-described NI35/250 and Semaphorin, a negative guidance factor. Specifically, the present inventors have paid their attention to the fact that NI35/250 has a growth-cone collapse activity and a neurite-growth inhibition activity in vitro (J. Neurosci., 8, 2381-2393 (1988); Science, 259, 80 (1993)), while known Semaphorins similarly possess a neurite-growth inhibition activity, and particularly Semaphorin III has also a growth-cone collapse activity. This suggested to the inventors the possibility that unknown Semaphorins which have not yet been identified may include the one having a function as a CNS-neuron regeneration inhibitor. Specifically, the present inventors"" idea was that, although Semaphorin, which is characterized in that 1) it is highly expressed in CNS of adult and 2) it is poorly expressed in fetus or peripheral tissues in adult where the neurite outgrowth is not inhibited, has not been identified yet, if one can identify a new unknown Semaphorin having such characteristics, the Semaphorin might function as a CNS-neuron regeneration inhibitor.
Thus, a DNA sequence encoding amino acids relatively well conserved among previously reported Semaphorin genes was firstly determined using EST (Expressed Sequence Tags) database, and as a consequence, a DNA fragment T08532(SEQ ID NO:20) was identified, which encodes, as a partial sequence, a sequence (Gln-Asp-Pro-Tyr-Cys-Gly-Trp-Ala) (SEQ ID NO:5)similar to that consisting of 8 amino acids highly conserved among Semaphorins (Gln (or Arg)-Asp-Pro-Tyr (or His)-Cys-Ala (or Gly)-Trp-Asp) (SEQ ID NO:6).
This T08532(SEQ ID NO:20) sequence contained undetermined bases, and its open reading frame could not be determined. Furthermore, T08532(SEQ ID NO:20) almost never contained any sequence which is common to Semaphorins, with the exception of the above amino acid sequence. Therefore, it could not be concluded at that stage that T08532(SEQ ID NO:20) is part of the gene encoding xe2x80x9cSemaphorinxe2x80x9d. Furthermore, distribution of T08532(SEQ ID NO:20) in fetus and adult tissues was absolutely unclear, and it was utterly impossible to expect that T08532(SEQ ID NO:20) may be a part of the gene encoding a Semaphorin having a function as a xe2x80x9cCNS-neuron regeneration inhibitorxe2x80x9d.
Thus, a DNA primer was firstly synthesized on the basis of the sequence information of T08532(SEQ ID NO:20), and used in PCR reaction together with cDNAs prepared from human hippocampal cDNA library as a template to clone a region corresponding to T08532(SEQ ID NO:20) and determine the base sequence (SEQ ID NO:7). Using the fragment thus cloned, rat and human cDNA libraries were then screened. As a result, the rat and human genes cloned in this procedures proved to be a novel Semaphorin gene having a sequence characteristic to Semaphorins. We named this novel Semaphorin xe2x80x9cSemaphorin Zxe2x80x9d.
Subsequent analysis revealed that Semaphorin Z of the present invention is highly expressed in CNS in the adult, but scarcely expressed in other tissues except for spleen, and that its expression in embryos was considerably lower than that in the adult, demonstrating an expression distribution which is considered reasonable for xe2x80x9cCNS-neuron regeneration inhibitorxe2x80x9d.
In addition, the present inventors have found that Semaphorin Z of the present invention has an inhibitory effect on neurite outgrowth. Furthermore, it has been found that a gene having a sequence complementary to Semaphorin Z gene inhibits the expression of Semaphorin Z.
Semaphorin Z of the present invention appears to be a CNS-neuron regeneration inhibitor in the adult, since it is highly expressed in CNS and inhibits neurite outgrowth as described above. The use of Semaphorin Z permits carrying out a screening to obtain Semaphorin Z inhibitor, and the inhibitor found by such screening system will be able to promote regeneration of CNS-neuron. Furthermore, since a gene having a sequence complementary to Semaphorin Z gene has inhibited the expression of Semaphorin Z as described above, such complementary gene may be used in a therapy for regeneration of CNS-neuron.
In addition, in view of the fact that Semaphorin Z of the present invention inhibits neurite outgrowth as described above, it may be used as a therapeutic or diagnostic agent for pains or immune diseases such as atopic dermatitis, by administering to peripheral tissues, which results in the inhibition of neurite outgrowth of PNS-neuron. Furthermore, since Semaphorin Z is a novel Semaphorin belonging to the Semaphorin family, it serves as an important research material or a laboratory reagent.
The present invention has been completed on the basis of the above findings.
That is, the gist of the present invention is as follows:
(1) Semaphorin Z DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or 3;
(2) Semaphorin Z open reading frame comprising the nucleotide sequence shown in residues 19 through 2682 of SEQ ID NO:1 or residues 39 though2702of SEQ ID NO: 3;
(3) Semaphorin Z protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 4;
(4) DNA which encodes a protein having Semaphorin domain and which hybridizes under stringent conditions to DNA comprising the nucleotide sequence shown in SEQ ID NO: 7;
(5) a protein encoded by the DNA of the above item (4);
(6) DNA which encodes a protein inhibiting neurite outgrowth and which hybridizes under stringent conditions to the DNA of the above item (1);
(7) DNA of the above item (6) which encodes a protein inhibiting neurite outgrowth of CNS-neuron;
(8) a protein encoded by the DNA of the above item (6) or (7);
(9) DNA which encodes a protein inhibiting neurite outgrowth, said protein containing insertions, deletions or substitutions of one or more amino acids in the protein of the above item (3);
(10) DNA of the above item (9) which encodes a protein inhibiting neurite 5 outgrowth of CNS-neuron;
(11) a protein encoded by the DNA of the above item (9) or (10);
(12) DNA which encodes a protein promoting neurite outgrowth of CNS-neuron, said protein containing insertions, deletions, or substitutions of one or more amino acids in the protein of the above item (3);
(13) a protein encoded by the DNA of the above item (12);
(14) DNA which is cloned from a human cDNA or genomic library, and which hybridizes under stringent conditions to DNA comprising at least part of the DNA of the above item (1) or at least part of the complementary strand thereof;
(15) an expression plasmid expressing one of the DNAs of the above items (1), (2), (4), (6), (7), (9), (10), (12), or (14);
(16) a transformant transformed with the expression plasmid of the above item (15);
(17) a process for producing a recombinant protein, said process being characterized in that it comprises culturing the transformant of the above item (16) under conditions in which the expression plasmid of the above item (15) can be expressed;
(18) a polypeptide comprising at least 6 amino acids of one of the proteins of the above items (3), (5), (8), (11), or (13);
(19) a polypeptide of the above item (18) which promotes neurite outgrowth of CNS-neuron;
(20) a polypeptide of the above item (18) characterized in that it contains aspartic acid residue at position 203 of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid residue corresponding to the position of said aspartic acid residue;
(21) DNA or RNA comprising 8 or more bases, or a chemically modified variant thereof, which has a sequence complementary to one of the DNAs of the above items (1), (4), (6), (7), or (14),;
(22) DNA or RNA of the above item (21), or a chemically modified variant thereof, characterized in that it inhibits an expression of one of the proteins of the above items (3), (5), or (8);
(23) an antibody against one of the proteins of the above items (3), (5), (8), (11), or (13), or against one of the polypeptides of the above items (18)-(20);
(24) a screening method for Semaphorin Z inhibitor, which method is characterized in that it employs one of the proteins of the above items (3), (5), (8), or (11);
(25) Semaphorin Z inhibitor obtained by the screening method of the above item (24);
(26) Semaphorin Z inhibitor of the above item (25) which comprises the protein of the above item (13), the polypeptide of the above item (19) or (20), or the antibody of the above item (23);
(27) a CNS-neuron regeneration promoter which is characterized in that it contains at least one of the DNAs or RNAs of the above item (22) or chemically modified variants thereof, or Semaphorin Z inhibitors of the above item (25) or (26);
(28) a neurite outgrowth inhibitor for PNS-neuron which is characterized in that it contains at least one of the proteins of the above items (3), (5), (8), or (11); and
(29) a transgenic animal in which one of the DNAs of the above items (1), (4), (6), (7), (9), (10), or (12) has been artificially inserted into its chromosome, or has been knocked out.
The 1st embodiment of the present invention is cDNA for rat Semaphorin Z which comprises the base sequence shown in SEQ ID NO: 1 or cDNA for human Semaphorin Z which comprises the base sequence shown in SEQ ID NO: 3. These DNAs, as described in Example 1, may be cloned by screening a cDNA library derived from CNS tissue using a DNA having the sequence shown in SEQ ID NO: 7 as a probe. Particular techniques for such cloning may be found in a standard text such as xe2x80x9cMolecular Cloning, 2nd ed.xe2x80x9d, Cold Spring Harbor Laboratory Press (1989). The nucleotide sequence of the cloned DNA may be determined by conventional methods, for example, using a sequence kit commercially available.
Alternatively, after publication of the nucleotide sequences of rat and human Semaphorin Z cDNAs of the present invention, one skilled in the art can also easily clone the rat and human Semaphorin Z gene in full length using part of said cDNA as a probe or primer, without using the cloning method as described above.
The 2nd embodiment of the present invention is an open reading frame of rat Semaphorin Z gene which comprises the nucleotide sequence shown in residues 19 through 2682 of SEQ ID NO:1 or an open reading frame of human Semaphorin Z gene which comprises the base sequence shown in residues 39 through 2702 of SEQ ID NO:3.
The 3rd embodiment of the present invention is a rat Semaphorin Z protein (referred to hereinafter simply as rat Semaphorin Z) which comprises the amino acid sequence shown in SEQ ID NO: 2 or a human Semaphorin Z protein (referred to hereinafter simply as human Semaphorin Z) which comprises the amino acid sequence shown in SEQ ID NO: 4.
Semaphorin Z contains Semaphorin domain characteristic to Semaphorins and this domain corresponds to a region extending from position 49 to position 580 of the amino acid sequence shown in SEQ ID NO: 2 or a region extending from position 48 to position 578 of the amino acid shown in SEQ ID NO: 4.
Semaphorin Z also contains a signal sequence at its N-terminal and this sequence is presumed to correspond to a region from position 1 to position 26 of the amino acid sequence shown in SEQ ID NO: 2 or from position 1 to position 25 of the amino acid sequence shown in SEQ ID NO: 4. The signal sequence is removed by processing during its transfer to membrane.
Preparation of Semaphorin Z may be achieved, for example, by linking a cloned Semaphorin Z cDNA to a known expression vector such as pET or pCDM8, and introducing the vector into an appropriate host cell to express and produce Semaphorin Z. The host cell may be procaryotic or eukaryotic. For example, Escherichia coli strains or animal cell lines are already conventionally used for such purpose and they are commercially available. Examples of animal host cells include COS-1, COS-7, CHO cells and the like.
To transform an appropriate animal host cell with an expression plasmid, a known procedure such as the DEAE-dextran method (Current Protocols in Molecular Biology, F. M. Ausubel et al.ed., John Wiley and Sons (1987)) may be used. As demonstrated in Example 7, Semaphorin Z of the present invention is localized in the cell membrane fraction which contains a sufficient amount of Semaphorin Z to be directly used in various assays. Therefore, various assays for Semaphorin Z activity may easily be conducted using the cell membrane fraction.
The cell membrane fraction may easily be prepared by homogenizing Semaphorin Z-expressing cells, isolating and purifying the fraction by centrifugation as described hereinafter in Example 7.
Semaphorin Z may be purified by, for example, an affinity purification using an antibody against Semaphorin Z described hereinafter in the section of the 23rd embodiment of the present invention, or conventional column chromatography.
The 4th embodiment of the present invention is a DNA which encodes a protein having semaphorin domain and which hybridizes under stringent conditions to DNA comprising the nucleotide sequence shown in SEQ ID NO: 7.
In the above description, xe2x80x9cDNA comprising the nucleotide sequence shown in SEQ ID NO: 7xe2x80x9d refers to a fragment cloned from cDNA by PCR reaction using the sequence information of the DNA xe2x80x9cT08532xe2x80x9d which encodes, in a part, a sequence (Gln-Asp-Pro-Tyr-Cys-Gly-Trp-Ala SEQ ID NO: 5) similar to the eight amino-acid sequence well conserved among Semaphorins (Gln (or Arg)-Asp-Pro-Tyr (or His)-Cys-Ala (or Gly)-Trp-Asp SEQ ID NO: 6). The DNA fragment corresponds to a region from position 1510 to position 1685 in the nucleotide sequence of rat Semaphorin Z shown in SEQ ID NO: 1, or a region from position 1524 to position 1699 in the nucleotide sequence of human Semaphorin Z shown in SEQ ID NO: 3.
As used herein, DNA which xe2x80x9chybridizes under stringent conditionsxe2x80x9d refers to such a DNA that hybridizes to DNA of SEQ ID NO: 7, for example, when hybridized under the following conditions: a formamide concentration of about 45% (v/v), a salt concentration of about 5xc3x97SSPE, and a temperature of about 42xc2x0 C., and washed under the following conditions: a salt concentration of 2xc3x97SSPE, and a temperature of about 42xc2x0 C., as described in Example 1.
Cloning of these DNAs is achieved by, for example, hybridization with DNA of SEQ ID NO: 7, and specifically may be carried out, for example, according to the procedures described in TINS, 15, 319-323 (1992) and references cited therein, and more specifically according to the following procedures.
That is, the cloning may be achieved by screening a cDNA or genomic library prepared from one of various animal tissues using DNA consisting of the nucleotide sequence shown in SEQ ID NO: 7 as a probe. The screening may be carried out according to, for example, the procedures as described in Example 1. Preferred cDNA libraries are those derived from an adult tissue of CNS, and a cDNA library derived from hippocampus, corpus striatum, or cerebellum is more preferred. As described above, the conditions shown in Example 1 or those described in TINS, 15, 319-323 (1992) and references cited therein may be used for the hybridization.
The DNA of the 4th embodiment of the present invention is also xe2x80x9cDNA which encodes a protein having semaphorin domainxe2x80x9d. As used herein, xe2x80x9csemaphorin domainxe2x80x9d refers to a domain consisting of 300-600 amino acid residues more than 20% of which are identical to those amino acid residues constituting semaphorin domain of one of 10 known Semaphorins (G-Sema I, T-Sema, I, D-Sema II, H-Sema III, C-Collapsin, Sem A, Sem B, Sem C, Sem D, Sem E) described in, for example, Cell, 75, 1389-1399 (1993) or Neuron, 14, 941-948 (1995). Those proteins having semaphorin domain more than 30% of which amino acids are identical to those amino acids of one of the known Semaphorins are particularly preferred. The identity of amino acids is determined by comparison using, for example, DNASIS Ver. 2.0 (HITACH Software Engineering) under conditions of ktup=1 and cutoff=1. More preferred proteins are those in which 10 or more cysteines, particularly 12 or more cysteines, of the 13 cysteines conserved in Semaphorin domains of the 10 known Semaphorins (for example, those cysteines marked in FIG. 1 on page 942 of Neuron, 14, 941-948 (1995)) are conserved.
Specific examples of DNA of the 4th embodiment of the present invention may include unknown Semaphorin genes which hybridizes under stringent conditions to DNA comprising the nucleotide sequence shown in SEQ ID NO: 7, including all the Semaphorin Z genes of mammal and avian. Between mammals or between mammal and avian, homologous genes have quite similar sequences, and usually more than 75%, in many cases more than 90%, of the base sequence are common each other. Therefore, all the Semaphorin Z genes of mammal and avian are included within the 4th embodiment of the present invention.
The 5th embodiment of the present invention is a protein encoded by DNA of the 4th embodiment of the present invention. Specifically, this embodiment is a protein which is encoded by DNA hybridizing under stringent conditions to DNA comprising the nucleotide sequence shown in SEQ ID NO: 7, and contains Semaphorin domain. These proteins can be expressed and purified by the methods similar to those used for a protein of the 3rd embodiment of the present invention.
These DNAs of the 4th embodiment of the present invention and the proteins of the 5th embodiment of the present invention can be achieved thanks to the discovery of Semaphorin Z which forms the core of the present invention. Once Semaphorin Z has been discovered, one can easily clone DNA of the 4th embodiment of the present invention and express a protein of the 5th embodiment of the present invention, according to conventional methods as described above. Therefore, DNAs of the 4th embodiment of the present invention and proteins of the 5th embodiment of the present invention both of which are found concomitantly with the discovery of Semaphorin Z also retain the essence of the present invention, and are thus included within the scope of the present invention.
The 6th embodiment of the present invention is DNA which encodes a protein inhibiting neurite outgrowth and which hybridizes under stringent conditions to DNA of the 1st embodiment of the present invention (rat and human Semaphorin Z DNA).
The DNA mentioned above hybridizes to DNA shown in SEQ ID NO: 1 or 3, and can be cloned, for example, by screening a cDNA or genomic library prepared from one of various animal tissues using DNA shown in SEQ ID NO: 1 or 3 as a whole or in part as a probe. Particular methods for screening and the like may be similar to those used for DNA of the 4th embodiment of the present invention. The xe2x80x9cstringent conditionsxe2x80x9d used herein may also be similar to those used for DNA of the 4th embodiment of the present invention.
The phrase xe2x80x9cinhibiting neurite outgrowthxe2x80x9d means that the protein has a collapse activity on growth cone of neuron as demonstrated in Example 8, or that the protein has a neurite-outgrowth inhibition activity. These activities may be measured using, for example, an expression product which is obtained by expressing said DNA by the methods similar to those used for expressing a protein of the 3rd embodiment of the present invention, and, for example, in the following manner:
Since Semaphorin Z is a membrane protein as confirmed in Example 7, activities of Semaphorin Z can easily be measured by using, as a test material, a membrane fraction of cells transformed with Semaphorin Z gene (see Example 8).
Activities of Semaphorin Z can be measured by various methods, and representative methods include, for example, those for a collapse activity on growth cone of neuron (M. Igarashi et al., Science, vol. 259, pp. 77-79 (1993)) or a neurite-outgrowth inhibition activity (J. A. Davies et al., Neuron, vol. 2, pp. 11-20 (1990); M. Bastmeyer, J. Neurosci., vol. 11, pp. 626-640 (1991)). A method of measuring a growth-cone collapse activity is described in detail in the paper (M. Igarashi et al., Science, vol. 259, pp. 77-79 (1993)). Briefly, the measurement may be carried out by a method in which cells expressing Semaphorin Z is homogenized, and the homogenate containing the cell membrane fraction or the purified membrane fraction is used (E. C. Cox et al., Neuron, vol. 2, pp. 31-37 (1990)), or by a method in which a protein extracted from the membrane fraction is reconstituted in a liposome and used as a test material (C. E. Bandtlow, Science, vol. 259, pp. 80-84 (1993)). To measure a growth-cone collapse activity using these materials, Semaphorin Z protein in one of the foregoing forms is added to neurons cultured under usual conditions (see, for example, xe2x80x9cCulturing, Nerve Cellsxe2x80x9d edited by Banker et al., MIT Press (1991)) in a container coated with a substance promoting the neurite outgrowth and the growth-cone formation, such as laminin, collagen, polylysine or polyornithine. When sufficient time has passed to occur a collapse of growth cone (typically from 30 minutes to one hour after the addition), those neurons are fixed with 1% glutaraldehyde or the like, and the number of the growth cones which have been collapsed is counted under a microscope. In this measurement, it is important that another sample is used as a control, which is prepared from cells not expressing Semaphorin Z according to the completely same procedures as those used for Semaphorin Z-expressing cells. Typically, normalization of the samples is conducted on the basis of the total amounts of protein included within the samples. To measure a neurite-outgrowth inhibition activity, part of the surface of a micropore filter or a culture container made of glass or plastics is coated with Semaphorin Z prepared as described above. The activity may be indicated, for example, by the inability of neurons cultured under usual conditions to adhere to the coated area, or a remarkable decrease in the rate of neurite outgrowth on the coated area, or the inability of invasion of growing neurites from the outside of the coated area into the coated area because of its stopping on the border between the coated and non-coated areas or its avoidance from the coated area. When a cluster of cells expressing Semaphorin Z is co-cultured with neurons in a collagen gel, the inability of outgrowing neurite to enter the cluster of cells expressing Semaphorin Z may also be used as an indicator (A. Sophia et al., Cell, vol. 81, 621-629 (1995)).
Specific examples of such DNAs of the 6th embodiment of the present invention, as well as examples of DNAs of the 7th embodiment of the present invention, may include, for example, all the Semaphorin Z genes of mammal and avian.
The 7th embodiment of the present invention is DNA of the 6th embodiment of the present invention which encodes a protein inhibiting neurite outgrowth of CNS-neuron.
In this context, the phrase xe2x80x9cinhibiting neurite outgrowth of CNS-neuronxe2x80x9d means that the protein has the activity of Semaphorin Z of the present invention, and this activity may be measured by using CNS-neuron as a cell for assay in the measurement described above in connection with the 6th embodiment of the present invention.
As described in the xe2x80x9cPrior Artxe2x80x9d section, CNS in adult mammals naturally contains a large amount of regeneration (outgrowth) inhibitor. It is, therefore, extremely difficult to measure in vivo an inhibitory effect on neurite outgrowth of CNS-neuron, and such inhibitory effect is usually measured by an in vitro method as described above in connection with the 6th embodiment of the present invention. Since these in vitro methods each have an individual characteristic, it is preferred to use more than one method to confirm the activity. Although preferred neurons used for a measurement of the activity are CNS-neurons such as spinal cord or motor neuron in motor cortex, PNS-neurons in superior cervical ganglion and dorsal root ganglion (DRG) may also be used because NI35/250 known as a CNS-neuron regeneration inhibitor has proved to have neurite-growth inhibition and growth-cone collapse activity also on these PNS-neurons (J. Cell Biol., 106, 1281-1288 (1988), Science, 259, 80-83 (1993)).
The 8th embodiment of the present invention is a protein encoded by DNA of the 6th or 7th embodiment of the present invention. Specifically, it is a protein which is encoded by DNA hybridizing under stringent conditions to DNA of the 1st embodiment of the present invention and which protein inhibits neurite outgrowth or which protein inhibits neurite outgrowth of CNS-neuron. These proteins can be expressed and purified by the methods similar to those used for a protein of the 3rd embodiment of the present invention. The activity may be measured by the methods described above in connection with the 6th and 7th embodiments of the present invention.
DNAs of the 6th and 7th embodiments of the present invention and the proteins of the 8th embodiment of the present invention can be achieved entirely thanks to the discovery of Semaphorin Z which forms the core of the present invention. Once Semaphorin Z has been found, one can easily clone and express DNA of the 6th or 7th embodiment of the present invention by conventional methods as described above. The protein of the 8th embodiment of the present invention having said activity can be then identified by subjecting the expression product thus obtained to an activity measurement system as described above. Therefore, DNAs of the 6th and 7th embodiments of the present invention and the proteins of the 8th embodiment of the present invention, both of which are easily found concomitantly with the discovery of Semaphorin Z, also retain the essence of the present invention, and are thus included within the scope of the present invention.
The 9th embodiment of the present invention is DNA which encodes a protein inhibiting neurite outgrowth, said protein containing insertions, deletions, or substitutions of one or more amino acids in the rat and human Semaphorin Zs of the 3rd embodiment of the present invention. The 10th embodiment of the present invention is DNA of the 9th embodiment of the present invention which encodes a protein inhibiting neurite outgrowth of CNS-neuron.
In this connection, one skilled in the art can easily introduce xe2x80x9can insertion, deletion, or substitution of one or more amino acidsxe2x80x9d by, for example, a site-directed mutagenesis (Methods in Enzymology, 100, 448xe2x80x94(1993)) or a PCR method (Molecular Cloning, 2nd ed., Chapter 15, Cold Spring Harbor Laboratory Press (1989), xe2x80x9cPCR A Practical Approachxe2x80x9d IRL Press, 200-210 (1991)). The inhibitory effect on neurite outgrowth can be measured by the methods described above in connection with the 6th and 7th embodiments of the present invention.
Based on the structural comparison of known Semaphorins, most of the conserved amino acids are located in Semaphorin domain, suggesting that these conserved amino acids are essential for expression of the activity of Semaphorin. Furthermore, the present inventors has found that a modified Sema III protein in which the aspartic acid residue at position 198 in its Semaphorin domain has been substituted with glycine does not have a growth-cone collapse activity (see Reference example 1 below). Accordingly, the aspartic acid at position 198 of Sema III is believed essential for expression of the activity. The amino acid residues corresponding to this position are highly conserved in known Semaphorins, and they are all aspartic acid with a few exceptions in which glutamic acid is located at this position. It is, therefore, believed that the amino acid residue at this position is also essential for expression of the activity of Semaphorins other than Sema III. In Semaphorin Z of the present invention, the amino acid residue corresponding to the position 198 of Sema III is presumed to be the aspartic acid at position 204 in the amino acid sequence of rat Semaphorin Z shown in SEQ ID NO: 3 or the aspartic acid at position 203 in the amino acid sequence of human Semaphorin Z shown in SEQ ID NO: 4.
Considering the above information, it is desirable to introduce insertions, deletions, or substitutions of one or more amino acids into the amino acid sequence not containing the residues conserved among Semaphorins, so as to retain the activity of Semaphorin Z in the modified protein (a protein encoded by DNA of the 9th or 10th embodiment of the present invention). Particularly, it is desirable not to modify the aspartic acid at position 204 in rat Semaphorin Z shown in SEQ ID NO: 2 and the aspartic acid at position 203 in human Semaphorin Z. In order to substitute an amino acid conserved among Semaphorins while retaining the activity of Semaphorin Z, it is desirable to substitute an amino acid having a similar side chain for the amino acid to be substituted. By substituting such amino acid having a similar side chain for a conserved amino acid, it may be possible to produce a modified protein which has an enhanced activity of Semaphorin Z. Such modified protein having an enhanced activity is highly suitable as a neurite-outgrowth inhibitor for PNS-neuron as will be described below in connection with the 28th embodiment of the present invention.
In the above-noted embodiment, xe2x80x9ca conserved amino acidxe2x80x9d refers to an amino acid located at a position at which more than 50% of Semaphorin genes shown in FIG. 2 of Cell, 75, 1389-1399 (1993) or FIG. 1 of Neuron, 14, 941-948 (1995) share the same amino acid.
The 11th embodiment of the present invention is a protein encoded by DNA of the 9th or 10th embodiment of the present invention. Specifically, the protein is a so-called xe2x80x9cmodified proteinxe2x80x9d which contains insertions, deletions, or substitutions of one or more amino acid in a protein of the 3rd embodiment of the present invention, and which inhibits neurite outgrowth or which inhibits neurite outgrowth of CNS-neuron. These proteins can be expressed and purified by the methods similar to those used for the protein of the 3rd embodiment of the present invention. The activity may be measured by the methods described above in connection with the 6th and 7th embodiments of the present invention.
DNAs of the 9th and 10th embodiments of the present invention and the proteins of the 11th embodiment of the present invention can be achieved entirely thanks to the discovery of Semaphorin Z which forms the core of the present invention. Once Semaphorin Z has been found, one can introduce therein insertions, deletions, or substitutions of one or more amino acids by the conventional methods as described above, and one can identify the protein of the 11th embodiment of the present invention by subjecting the modified protein thus obtained to an activity measurement system as described above. Therefore, DNAs of the 9th and 10th embodiments of the present invention and the proteins of the 11th embodiment of the present invention, both of which are easily attained concomitantly with the discovery of Semaphorin Z, also retain the essence of the present invention, and are thus included within the scope of the present invention.
The 12th embodiment of the present invention is DNA encoding a protein which contains insertions, deletions, or substitutions of one or more amino acids in rat or human Semaphorin of the 3rd embodiment of the present invention, and which protein promotes neurite outgrowth of CNS-neuron.
The insertion, deletion, and substitution in these DNAs can be introduced therein according to the procedures similar to those used for DNA of the 9th embodiment of the present invention. The activity which promotes neurite outgrowth of CNS-neuron can easily be measured by, for example, adding a test material (i.e., a modified Semaphorin Z protein as a candidate) to an assay system for the activity described above in connection with the 6th and 7th embodiments of the present invention. For details, see the descriptions of the 24th embodiment of the present invention.
A specific example of these proteins may be a modified Semaphorin Z protein of which neurite-outgrowth inhibition activity on CNS-neuron has been inactivated. When the modified protein which does not have such inhibition activity binds to a receptor for Semaphorin Z or to Semaphorin Z itself, the neurite-outgrowth promotion effect on CNS-neuron will arise. As described above in connection with the 9th embodiment of the present invention, it has been suggested that the active site of Semaphorin may be located in Semaphorin domain, and particularly, it may be located at the aspartic acid at position 204 in rat Semaphorin Z or the aspartic acid at position 203 in human Semaphorin Z. Accordingly, in order to eliminate the Semaphorin Z activity, it is desirable to conduct insertions, deletions or substitutions of one or more amino acids at the conserved amino acid(s) in said Semaphorin domain, preferably directed to the aspartic acid at position 204 in rat Semaphorin Z or to the aspartic acid at position 203 in human Semaphorin Z. In such cases, those substitutions in which an amino acid having a side chain of a distinct nature is substituted for the original amino acid are desirable.
Since the protein encoded by DNA of the 12th embodiment of the present invention promotes neurite outgrowth of CNS-neuron as described above, it serves as a regeneration promoter for CNS-neuron as described below in connection with the 27th embodiment of the present invention.
The 13th embodiment of the present invention is a protein encoded by DNA of the 12th embodiment of the present invention. Specifically, it is a protein which contains insertions, deletions, or substitutions of one or more amino acids in the protein of the 3rd embodiment of the protein invention, and which promotes neurite outgrowth of CNS-neuron. These proteins can be expressed and purified by the methods similar to those used for the protein of the 3rd embodiment of the present invention. The neurite-outgrowth promotion effect on CNS-neuron may be measured by the methods described above in connection with the 12th embodiment of the present invention.
These DNAs of the 12th embodiment of the present invention and the proteins of the 13th embodiment of the present invention can be achieved entirely thanks to the discovery of Semaphorin Z which forms the core of the present invention. Once Semaphorin Z has been found, one can produce a modified protein in which insertions, deletions, or substitutions have been introduced by the conventional methods as described above, and one can easily identify the modified protein having a neurite-outgrowth promotion activity by subjecting it to a measurement system (screening system) for such activity as described above. DNAs of the 12th embodiment of the present invention and the proteins of the 13th embodiment of the present invention, both of which are easily attained concomitantly with the discovery of Semaphorin Z, also retain the essence of the present invention, and are thus included within the scope of the present invention.
The 14th embodiment of the present invention is DNA which is cloned from a human cDNA or genomic library and which hybridizes under stringent conditions to DNA comprising at least part of rat or human Semaphorin Z DNA of the 1st embodiment of the present invention or at least part of the complementary strand thereof.
Methods of Cloning are described in detail in, for example, xe2x80x9cMolecular Cloning 2nd ed.xe2x80x9d, Cold Spring Harbor Laboratory Press (1989), and specifically include, for example, methods employing hybridization or PCR reaction. Although a preferred library used herein is a genomic library derived from human, a cDNA library derived from CNS-neuron in the adult may also be used. Those methods employing hybridization may be carried out according to, for example, TINS, 15, 319-323 (1992) and references cited therein. Those methods employing PCR may be carried out according to, for example, xe2x80x9cPCRxe2x80x9d, edited by McPherson et al. ed., 1991, IRL Press.
The DNA thus cloned is a gene for human Semaphorin Z, and such DNAs include not only the full length DNA but also its DNA fragments comprising more than 200 bases. Specific examples of DNA of the 14th embodiment of the present invention may include chromosomal DNAs containing 5xe2x80x2 and/or 3xe2x80x2 transcriptional control regions, noncoding regions of exons, introns, or the like, in addition to those consisting of a region encoding amino acids. Such sequences which do not encode any amino acids are also quite useful, for example, when it is desired to develop a medicine using antisense techniques described below.
Since these DNAs of the 14th embodiment of the present invention are also easily achieved concomitantly with the discovery of Semaphorin Z, it goes without saying that they are included within the scope of the present invention.
The 15th embodiment of the present invention is an expression plasmid expressing one of DNAs of the 1st, 2nd, 4th, 6th, 7th, 9th, 10th, 12th, and 14th embodiments of the present invention. The 16th embodiment of the present invention is a transformant transformed with said expression plasmid. The 17th embodiment of the present invention is a process for producing a recombinant protein which process is characterized in that it comprises culturing said transformant under conditions in which said expression plasmid can be expressed. As described above in connection with the 3rd embodiment of the present invention, methods of preparing an expression plasmid and a transformant, and methods of producing a recombinant protein, per se, are all well known to those skilled in the art.
The 18th embodiment of the present invention is a polypeptide comprising at least 6 amino acids of one of the proteins of the 3rd, 5th, 8th, 11th, and 13th embodiments of the present invention. In this connection, the limitation xe2x80x9cat least 6 amino acidsxe2x80x9d is based on the fact that a minimal size of polypeptide capable of forming a stable structure consists of 6 amino acids, and preferred polypeptides are those consisting of 10-20 amino acids. A short polypeptide such as those consisting of 10-20 amino acids can be synthesized on a peptide synthesizer, while a longer polypeptide can be obtained by preparing DNA through usual genetic engineering, and expressing it in, for example, an animal cell as described above. The polypeptide thus prepared can also be modified by usual methods.
These polypeptides can be applied to medicaments as described below in connection with the 19th and 20th embodiments of the present invention and can also be used for producing antibodies.
The 19th embodiment of the present invention is a polypeptide of the 18th embodiment of the present invention which promotes neurite outgrowth of CNS-neuron. Such polypeptide may be prepared by the methods described above in connection with the 18th embodiment of the present invention. The promotion effect on neurite outgrowth of CNS-neuron can easily be measured as described above in connection with the 12th embodiment of the present invention by adding a test substance (i.e., a polypeptide derived from Semaphorin Z as a candidate) to an activity measurement system described above in connection with the 6th and 7th embodiments of the present invention. For details, see the descriptions of the 24th embodiment of the present invention.
A specific example of these polypeptides may be a Semaphorin Z polypeptide of which neurite-outgrowth inhibition activity on CNS-neuron has been lost. When a polypeptide which does not have such inhibition activity bind to a receptor for Semaphorin Z or to Semaphorin Z itself, the neurite-outgrowth promotion effect on CNS-neuron will arise. As described below in connection with the 27th embodiment of the present invention, such polypeptide may serve as a CNS-neuron regeneration promoter.
The 20th embodiment of the present invention is a polypeptide of the 18th embodiment of the present invention characterized in that it contains aspartic acid residue at position 203 of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid corresponding to the position of said aspartic acid residue. Such polypeptide may be prepared by the methods described above in connection with the 18th embodiment of the present invention.
As described above in connection with the 9th embodiment of the present inventions, the aspartic acid residue at position 203 of human Semaphorin Z shown in SEQ ID NO: 4 (in the case of rat, the aspartic acid at position 204) seems essential for expression of the activity of Semaphorin Z. Since this amino acid may possibly be involved in the binding between Semaphorin Z and Semaphorin Z receptor, a polypeptide of the 20th embodiment of the present invention containing this amino acid residue may interfere with the neurite-outgrowth inhibition activity on CNS-neuron exerted by Semaphorin Z, by binding to the receptor for Semaphorin Z or to Semaphorin Z itself, resulting in promotion of neurite outgrowth of CNS-neuron. A polypeptide having such effect may serve as a CNS-neuron regeneration promoter as described below in connection with the 27th embodiment of the present invention. Such neurite-outgrowth promotion activity on CNS-neuron can easily be measured as described above in connection with the 12th embodiment of the present invention by adding a test substance (i.e., a polypeptide derived from Semaphorin Z polypeptide as a candidate) to an activity measurement system described above in connection with the 6th and 7th embodiments of the present invention. For details, see the descriptions of the 24th embodiment of the present invention.
In the above-noted embodiment, xe2x80x9can amino acid corresponding to the position of said aspartic acidxe2x80x9d refers to an amino acid which is located at the position corresponding to position 203 in human Semaphorin Z, when the amino acid sequence of the protein of the 5th, 8th, 11th, or 13th embodiment of the present invention is aligned with the amino acid sequence shown in SEQ ID NO: 4 so that a maximal identity is obtained. Accordingly, xe2x80x9ca polypeptide characterized in that it contains an amino acid corresponding to the position of said aspartic acidxe2x80x9d refers to a polypeptide which comprises such amino acid at the position corresponding to position 203 of human Semaphorin Z as well as a few amino acids contiguous to said amino acid.
The 21st embodiment of the present invention is DNA or RNA comprising 8 or more bases, or a chemically modified variant thereof, which has a sequence complementary to one of the DNAs of the 1st, 4th, 6th, 7th, and 14th embodiments of the present invention.
In this context, xe2x80x9cDNA or RNA which has a sequence complementary to . . . xe2x80x9d (referred to hereinafter as xe2x80x9cantisense nucleotidexe2x80x9d) is a so-called antisense oligonucleotide, antisense RNA, or antisense DNA, and it may be artificially prepared using a DNA synthesizer, or may be obtained by, for example, expressing a gene in the direction opposite to the usual case (i.e., in the antisense direction) as described below in Example 9. For details, see the descriptions of the 27th embodiment of the present invention.
These antisense nucleotides are used for inhibiting the expression of Semaphorin Z as described below in connection with the 22nd embodiment of the present invention. In addition, they are also useful as laboratory reagents for, for instance, in situ hybridization. In this embodiment, xe2x80x9ca chemically modified variantxe2x80x9d specifically refers to such a variant that is chemically modified so as to enhance the transferability of the antisense oligonucleotide into cells or the stability of the antisense oligonucleotide in the cells. Examples of such chemically modified variant are phosphorothioate, phosphorodithioate, alkylphosphotriester, alkyl phosphonate, alkyl phosphoamidate, and the like derivatives (xe2x80x9cAntisense RNA and DNAxe2x80x9d, WILEY-LISS, 1992, pp. 1-50, J. Med. Chem., 36, 1923-1937 (1993)). These chemically modified variant may be prepared according to, for example, the above-mentioned papers.
The 22nd embodiment of the present invention is DNA or RNA of the 21st embodiment of the present invention, or a chemically modified variant thereof, which is characterized in that it inhibits the expression of one of the proteins of the 3rd, 5th, and 8th embodiment of the present invention.
A mRNA produced by usual gene transcription is a sense-strand. The antisense nucleotides, that is, antisense oligonucleotide and antisense DNA or RNA, or chemically modified variants thereof can bind to the sense-strand mRNA in cells to inhibit the expression of that particular gene. Therefore, the above-described antisense nucleotides or chemically modified variants thereof can inhibit the expression of Semaphorin Z of, for example, the 3rd embodiment of the present E-invention, thereby inhibiting the activity of said Semaphorin Z. Antisense nucleotides or chemically modified variants thereof having such effect serve as CNS-neuron regeneration promoters as described below in connection with the 27th embodiment of the present invention.
It can easily be determined whether a particular antisense nucleotide prepared, or a chemically modified variant thereof has a desired inhibitory effect or not, by directly introducing the antisense oligonucleotide itself or, as will be described below in Example 11, introducing a gene which produces said antisense RNA when transcribed, into a cell expressing Semaphorin Z, and then determining whether the amount of the expressed Semaphorin Z is decreased or not.
Examples of an antisense nucleotide exhibiting such inhibitory effect may be those having sequences complementary to the coding region or the 5xe2x80x2 or 3xe2x80x2 noncoding region of Semaphorin cDNA of the above-described embodiments. Especially preferred are those having sequences complementary to the transcription initiation site, translation initiation site, 5xe2x80x2 noncoding region, exon-intron junction region, or 5xe2x80x2 CAP region. As described in Example 11, an antisense RNA for Semaphorin Z of the present invention has been confirmed to inhibit the expression of Semaphorin Z. Therefore, it may serve as a CNS-neuron regeneration promoter of the 27th embodiment of the present invention described below.
The 23rd embodiment of the present invention is an antibody against one of the proteins of the 3rd, 5th, 8th, 11th, and 13th embodiments of the present invention or against one of the polypeptides of the 18th-20th embodiments of the present invention. Such antibody can easily be produced by immunizing a mouse or rabbit against a recombinant Semaphorin Z protein of claim 17 or a peptide of claim 18, according to the procedures described in, for example, Current Protocols in Immunology, pp. 2.4.1-2.6.6 (1992, J. E. Coligan ed.). Monoclonal antibodies can also easily be produced by the methods described in the above-mentioned reference. Such antibodies may be used in affinity chromatographies or screening of cDNA libraries, and as a pharmaceutical or diagnostic agent, or a laboratory reagent. Some of such antibodies can neutralize the activity of Semaphorin Z. These neutralizing antibodies can easily be identified as described above in connection with the 12th embodiment of the present invention by adding a test substance (i.e., a candidate antibody against Semaphorin Z) to an activity measurement system described above in connection with the 6th and 7th embodiments of the present invention. As described below in connection with the 27th embodiment of the present invention, such neutralizing antibody may serve as a CNS-neuron regeneration promoter.
One skilled in the art can easily prepare the above-described polypeptide of the 18th-20th embodiments of the present invention, DNA or RNA of the 21st or 22nd embodiment of the present invention or chemically modified variant thereof, and the antibody of the 23rd embodiment of the present invention, only if Semaphorin Z has been discovered. In addition, as described above, the state of the art allows one skilled in the art to easily determine whether or not such substances have particular functions such as neurite-outgrowth promotion effect on CNS-neuron. Accordingly, these substances are all included within the scope of the present invention.
The 24th embodiment of the present invention is a screening method for Semaphorin Z inhibitors, which method is characterized in that it employs one of the proteins of the 3rd, 5th, 8th, and 11th embodiments of the present invention. As used herein, xe2x80x9cSemaphorin Z inhibitorxe2x80x9d refers to a substance which inhibits, for example, the neurite-outgrowth inhibition activity on CNS-neuron exerted by Semaphorin Z.
The screening is conducted by adding a test substance to a Semaphorin Z activity measurement system described above in connection with the 6th and 7th embodiments of the present invention. Specifically, inhibition of the Semaphorin Z activity resulted from the addition of the test substance to the culture medium throughout the incubation period or only temporarily in the incubation period can be used as an indicator in the Semaphorin Z activity measurement. It is also important to confirm that the test substance alone does not influence the survival and neurite-outgrowth of neurons at the same concentration. When both of these requirements are fulfilled, one can consider the test substance as a Semaphorin Z inhibitor. Although it is preferred to prepare in advance the test substance in the form of aqueous solution, an organic solvent such as DMSO may also be used as a solvent. In any cases, it is important to minimize the volume of the solvent so as to exclude any effects of the solvent on neurons. Specifically, the volume to be added should be less than an equal volume, preferably less than 1/10 volume, and more preferably less than 1/100 volume relative to the culture medium. Semaphorin Z inhibitor thus obtained may be used as a CNS-neuron regeneration promoter as described below in connection with the 27th embodiment of the present invention.
The 25th embodiment of the present invention is Semaphorin Z inhibitor which is obtained by the screening method of the 24th embodiment of the present invention. The inhibitor may have any structure and any form, provided that it inhibits the activity of Semaphorin Z.
The 26th embodiment of the present invention is Semaphorin Z inhibitor of the 25th embodiment which comprises the protein of the 13th embodiment of the present invention, the polypeptide of the 19th or 20th embodiment of the present invention, or the antibody of the 23rd embodiment of the present invention. Specifically, it comprises the protein of the 13th embodiment, the polypeptide of the 19th or 20th embodiment, or the antibody of the 23rd embodiment of the present invention which inhibits the activity of Semaphorin Z. The inhibitors can easily be selected by conducting the screening of the 24th embodiment of the present invention, and the inhibitors thus selected may serve as a CNS-neuron regeneration promoter as described below in the 27th embodiment of the present invention.
The 27th embodiment of the present invention is a CNS-neuron regeneration promoter characterized in that it comprises at least one of DNAs or RNAs of the 22nd embodiment of the present invention, or chemically modified variants thereof, or at least one of Semaphorin Z inhibitors of the 25th or 26th embodiment of the present invention. This embodiment relates to the use of substances for xe2x80x9cpromotion of CNS-neuron regenerationxe2x80x9d. The following descriptions explain the use, dose and the like, of the substances.
As described above in connection with the 22nd embodiment of the present invention, the antisense nucleotide of the 22nd embodiment or its chemically modified variant can inhibit the expression of Semaphorin Z gene. Accordingly, such antisense nucleotide may decrease the abundance of the Semaphorin protein, and promote the regeneration of CNS-neuron. Therapeutic methods using the nucleotide or the variant include those in which the antisense oligonucleotide or its chemically modified variant itself is administered, and those in which antisense RNA is produced in cells.
In the method in which the antisense oligonucleotide or its chemically modified variant itself is administered, a preferred antisense oligonucleotide has a length of, for example, about 8-200 bases, and more preferably 8-25 bases, and especially preferably 12-25 bases. Antisense oligonucleotide or its chemically modified variant may be formulated by mixing it with stabilizing agent, buffer, solvent and the like prior to its administration. Such formulation may be co-administered with, for example, an antibiotic, anti-inflammatory, or anesthetic agent. Although the formulation thus prepared may be administered via various routes, it is preferred to topically administered at a site in which neurons are notably disordered. Usually, the regeneration of neuron takes several days to several months, and the formulation is administered every day, or every several days to several weeks. To avoid such frequent administrations, a sustained-release mini-pellet formulation may be prepared and embedded near the affected site. Alternatively, a formulation may be gradually and continuously administered to the affected site by means of, for example, an osmotic pump. Typically, the dose is adjusted so that the concentration at the site of action will be 0.1 nM-10 xcexcM.
In the method in which an antisense RNA is produced in a cell, a preferred antisense RNA has a length of, for example, more than 100 bases, preferably more than 300 bases, and more preferably more than 500 bases.
The methods by which a gene expressing an antisense RNA is introduced into a patient include an in vivo method in which the gene is directly introduced into a cell in a living body, and an ex vivo method in which the gene is introduced into a particular cell ex vivo and the cell is returned into the body (Nikkei Science, April, 1994, pp. 20-45; Gekkan-Yakuji, 36 (1), 23-48 (1994); Jikkenn-Igaku-Zokan, 12 (15), 1994; and references cited therein). An in vivo method is more preferred.
Such in vivo methods include a method employing recombinant viruses and other methods (Nikkei Science, April, 1994, pp. 20-45; Gekkan-Yakuji, 36 (1), 23-48 (1994); Jikken-Igaku-Zokan, 12 (15), in its entirety (1994); and references cited therein).
The methods employing recombinant viruses may include the methods in which Semaphorin gene is incorporated into a virus genome of, for example, retrovirus, adenovirus, adeno-associated virus (AAV), herpesvirus, vaccinia virus, poliovirus, or sindbis virus, and the virus is introduced into a living body. Among these methods, those employing retrovirus, adenovirus or adeno-associated virus are particularly preferred.
Other methods may include a liposome method or a lipofectin method. The liposome method is particularly preferred.
The ex vivo methods which may be used include, besides those described above, a micro-injection method, a calcium phosphate method, electroporation and the like.
Administration of the gene to a patient is carried out via appropriate routes depending on particular disease or symptom to be treated, and the like. For example, it may be administered intravenously, intraarterially, subcutaneously, or intramuscularly, or directly administered into an affected site such as neuron. For example, when spinal cord is infected with the recombinant viruses, the expression of Semaphorin gene is inhibited exclusively in the spinal cord. Usually, the expression of antisense RNA lasts several days to several months, and such single infection is sufficient to allow the regeneration of neuron. When expressed insufficiently, the gene may be re-introduced. When administered by an in vivo method, the gene may be formulated in the form of, for example, a solution, and typically it is formulated in the form of an injection containing Semaphorin gene as an active ingredient to which conventional carrier and the like may be added, if necessary. In the case of liposomes or membrane-fused liposomes (such as Sendai virus (HVJ)-liposomes) containing Semaphorin gene, the liposome preparations may be in the form of a suspension, a frozen preparation, a centrifugally-concentrated frozen preparation or the like.
Although the amount of Semaphorin gene in the formulation may vary depending on the disease to be treated, the age and weight of the patient, and the like, it is typically 0.0001-100 mg, and preferably 0.001-10 mg, and such formulation is preferably administered once every several days to several months.
As described above in connection with 12th and 13th embodiments of the present invention, one can prepare a modified Semaphorin Z protein in which the neurite-outgrowth inhibition activity on CNS-neuron has been abolished. When administered into a living body, such modified protein may bind to receptors for Semaphorin Z or to Semaphorin Z itself, resulting in an inhibition of the Semaphorin Z activity and a promotion of the regeneration of CNS-neuron.
Such modified protein of Semaphorin Z is formulated with a stabilizer, buffer, and diluent, and administered to a patient for therapy. Such formulation may be administer by any one of various routes, and it is preferred to topically administer to the focal site. Since regeneration of neuron usually takes several days to several months, the formulation is administered once or more in order to continuously inhibit the Semaphorin Z activity throughout the period. When administered more than once, it is desirable to administer it every day or repeatedly at appropriate intervals. When administered to CNS by injection, for example, into spinal cord, several hundreds xcexcg to 2 g, preferably less than several tens mg, are used per administration. To reduce the administration frequency, it may be administered using a sustained-release formulation or gradually administered over a long period by means of, for example, an osmotic pump. Alternatively, it may be S administered by grafting into a living body a cell expressing such modified Semaphorin Z protein.
The peptide of the 19th or 20th embodiment of the present invention may suppress the inhibition activity of Semaphorin Z on neurite outgrowth of CNS-neuron by binding to receptors for Semaphorin Z, resulting in a promotion of regeneration of CNS-neuron. As described above in connection with the 20th embodiment of the present invention, polypeptides having such effect include, for example, a polypeptide characterized in that it contains the aspartic acid at position 203 of human Semaphorin Z shown in SEQ ID NO: 4 or an amino acid residue corresponding to said aspartic acid. The suppression may be any one of competitive, noncompetitive, uncompetitive, and allosteric inhibitions.
As for the methods of formulating or administering such polypeptides, and their doses, see the above section xe2x80x9c2) Modified protein of Semaphorin Zxe2x80x9d.
A neutralizing antibody which neutralizes the activity of Semaphorin Z may suppress, when administered into a living body, the activity of Semaphorin Z, and promote the regeneration of CNS-neuron.
The methods of formulating or administering such neutralizing antibody and their doses may be the same as described in the above section xe2x80x9c2) Modified protein of Semaphorin Zxe2x80x9d. Alternatively, a method in which cells producing a monoclonal antibody are grafted directly into CNS may also be used.
The above-described screening method of the 24th embodiment of the present invention can be established only after the discovery of Semaphorin Z, a representative protein of the present invention which inhibits neurite outgrowth of CNS-neuron. Once Semaphorin Z has been found, one can easily carry out the screening by using the method as described above. By carrying out such screening, one can easily select Semaphorin Z inhibitor of the 25th or 26th embodiment of the present invention which inhibits the neurite-outgrowth inhibition activity on CNS-neuron exerted by Semaphorin Z. Then, such inhibitor, or an antisense DNA or RNA, or its chemically modified variant, as described above which controls the expression of Semaphorin Z is used as a curative ingredient to formulate a CNS-neuron regeneration promoter of the 27th embodiment of the present invention. Therefore, these are all achieved concomitantly with the discovery of Semaphorin Z, and are all included within the scope of the present invention.
The 28th embodiment of the present invention is a neurite-outgrowth inhibitor for PNS-neuron characterized in that it contains at least one of the proteins of the 3rd, 5th, 8th and 11th embodiments of the present invention. Although the proteins of these embodiments inhibit the neurite outgrowth of CNS-neuron, they are also expected to inhibit the neurite outgrowth of PNS-neuron, since PNS-neuron also probably expresses a receptor for Semaphorin Z, and receptors for other Semaphorins also probably react with Semaphorin Z. Accordingly, they may serve as a therapeutic agent for pain or an immune disease such as atopic dermatitis, by virtue of their inhibition activity on neurite outgrowth of PNS-neuron.
As for the methods of formulating or administering such proteins, and their dose, see the above section xe2x80x9c2) Modified protein of Semaphorin Zxe2x80x9d.
The 29th embodiment of the present invention is a transgenic animal in which the DNA of the 1st, 4th, 6th, 7th, 9th, 10th or 12th embodiment of the present invention has been artificially inserted into its chromosome, or has been knocked out.
As apparent from the following references, one skilled in the art can quite easily produce a transgenic animal which has the gene of the 1st, 4th, 6th, 7th, 9th, 10th or 12th embodiment of the present invention inserted into its chromosome, on the basis of the gene information on Semaphorin Z of the present invention (Manipulation of Mouse Embryo, B. Hogan et al. ed., 1986, Cold Spring Harbor Laboratory; Shinichi Aizawa, Gene Targeting, 1995, Yodosha, etc.). Accordingly, the transgenic animal thus produced is naturally included within the scope of the present invention. The transgenic animal thus produced is very useful as an animal model for developing pharmaceuticals or an animal used for screening of pharmaceuticals. Furthermore, a so-called knockout animal in which the gene of the 1st, 6th or 7th embodiment of the present invention has been deleted at the chromosomal level is characterized in that it does not contain such gene. As described in literatures, or as apparent from the common knowledge in the art, such knockout animals cannot be produced without the gene information on Semaphorin Z of the present invention. It goes without saying, therefore, that such knockout animals are included within the scope of the present invention.
While Semaphorin Z plays an important role in inhibiting the neurite outgrowth of CNS-neuron in vivo as described above, it has been also suggested that Semaphorin gene may have other unknown functions such as immunosuppression (Cell, 75, 1389-1399 (1993)). Accordingly, it is quite important to investigate the expression of Semaphorin Z gene or the distribution and function of Semaphorin Z protein for studying this technical field or for diagnosing a patient, for example, with a neural disease. The present invention can provide a gene probe, an antibody, a recombinant protein, a transgenic animal and the like which are useful for such purposes.